The electron microscope uses a beam of accelerated electrons to create an image of the specimen. It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing the visualisation of much smaller objects in finer detail.
Immuno-electron microscopy (immunogold labelling of Tokuyasu cryosections)
In this variation of electron microscopy (EM), specimens are chemically fixed, cryo-protected and frozen. After this treatment, the sample is hard enough to be thin-sectioned by cryo-ultramicrotomy. The cryo-sections are then thawed and exposed to probes or antibodies. This technique is very sensitive and adapted to identifying membrane compartments in cells.
3D scanning electron microscopy (SEM) of plastic embedded samples
SEM’s can be used to automatically acquire serial images of plastic embedded biological samples. Three techniques currently fall under this category. The first one, identified as array tomography (AT), is when ribbons of serial sections are collected on conductive supports and imaged sequentially. The other two techniques involve imaging the surface of a resin-embedded sample. Upon ablation of a thin layer from this surface, a new image can be acquired. When iterated, this process leads to the automated acquisition of serial images from the sample. In general, there are two different ways for ablating the block surface, with an ion beam or with an integrated ultramicrotome. Respectively: focused-ion-beam scanning EM (FIB-SEM) and Serial Block Face Scanning EM (SBF-SEM or SBEM).
Ribbons of sections from a resin embedded block are collected by ultramicrotomy. Different techniques and hardware are available, with various degrees of automation leading to the collection of sections from very large samples (full organisms, large pieces of tissues in the mm range). Ribbons are collected on conductive substrates and transferred to the SEM for imaging.
Focused-ion-beam scanning EM (FIB-SEM)
A full understanding of the fine organization of cells and tissues requires their high-resolution visualization in three dimensions (3D). Plastic embedded cells can be imaged with automated focused ion beam (FIB) - scanning electron microscopy (SEM). In this technique, the FIB removes a thin slice from the block, after which the exposed face is imaged with the SEM. This process is repeated until the desired volume is imaged, allowing for 3D imaging at high resolution (sub 5 nm) in x, y, and z directions. This technique is especially suited to obtain ultrastructural information of subcellular regions or even an entire cell.
Serial Block Face Scanning EM (SBF-SEM or SBEM)
In this technique a miniaturized ultramicrotome, embedded in the chamber of the SEM, removes thin layers of the resin block (down to 25 nm) exposing a cross-section of the sample. The sequential imaging and sectioning enables automated acquisition of serial images. This method is particularly adapted for voluminous samples, such as small model organisms and tissues.
Serial section Transmission Electron Microscopy (ssTEM)
ssTEM is performed on series of thin sections that are acquired by conventional ultramicrotomy. The sections are collected on EM grids and imaged sequentially by transmission electron microscopy (TEM).
TEM tomography and STEM tomography
In TEM tomography thick plastic sections (typically 300nm) are placed on an EM grid and are tilted in the electron beam to +/- 70 degrees. At each tilt angle, a 2D image is acquired and the series of images are later computed to reconstruct the 3D volume: the tomogram. Serial thick sections can be imaged the same way, which then results in ssTEM-tomography. Complementary modalities, such as STEM tomography can deal with thicker sections (in the μm range).
CryoEM is currently not offered as open access technology in the EuBI technology portfolio.